Detection of specific chemical species in liquid solutions can be a complicated task in which an array of analytical equipment is used. In some instances, utilizing a marker, such as luminescent or radioactive markers which identify the target species, or analyte, can lack precision or sensitivity in some cases. Increasingly, mass spectrometry (MS) with an atmospheric pressure ionization (API) source is used by those seeking to quantify one or more analyte in a complex liquid solution. Mass spectrometric analysis requires that the sample be ionized, that is to say that the species in the sample have a mass and a net charge, either positive or negative. The atmospheric pressure ionization source converts charge-neutral analytes into ions in the gas phase that can be analyzed with mass spectrometry.
An atmospheric pressure ionization source can achieve ionization of species in a liquid sample in various ways. Some of the most common techniques are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). These two techniques have in common atomizing the sample by expelling the sample liquid through a narrow tube while heating the tube. Droplets of the sample liquid evaporate into the constituents of the sample, including the target chemical species. As the evaporated constituents of the sample travel from the narrow tube towards the mass spectrometer inlet, they travel through a large electrical potential and become ionized.
Mass spectrometric analysis is useful in quantifying medications or toxins in biological samples like blood, urine, or tissue extracts, as well as monitoring pesticides or pollutants in food or water. These types of sample solutions may contain high concentrations of salts or buffers, such as pH buffers, and these buffers make the ionization of analytes using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) highly inefficient. This effect is known as ion suppression and is the direct result of excess of salt becoming ionized instead of the analytes of interest. Currently, analysts using mass spectrometry (MS) fractionate complex samples prior to MS analysis, effectively separating out much of the analytes of interest from the salts or buffers prior to ionization. Fractionation is typically done by liquid chromatography (LC), most often high-pressure liquid chromatography (HPLC). Analysts use various chromatography media, solvents, additives and temperature to optimize the fractionation of analytes from interferents, such as salts and buffers. The direct coupling of high-pressure liquid chromatography (HPLC) fractionation to mass spectrometric (MS) analysis is very common and is known as high-pressure liquid chromatography-mass spectrometry (HPLC-MS).
Commercial systems for high-pressure liquid chromatography mass spectrometry (HPLC-MS) can be large, complicated systems. Such systems can include high-pressure liquid pumps, two or more solvent reservoirs, a solvent mixer to create the needed gradients in the solvent ratio, a valve or other sample introduction mechanism, a chromatography column for performing the fractionation, and a detector, which in the case of HPLC-MS is a mass spectrometry detector. The complexity of commercial HPLC-MS systems can vary and such systems may have the ability to perform at different pressures, may include automated sample introduction, may include temperature controls, and may include additional optical detectors, for example those capable of measuring light absorbance, fluorescence, or light scattering from a liquid sample.
When using a commercial high-pressure liquid chromatography-mass spectrometry (HPLC-MS) system, the user should have some degree of skill or knowledge to obtain accurate and reliable results. The user optimizes each analysis through multiple decisions, such as by selecting the correct chromatography media and solvents, though other variables can be controlled, such as temperature, pressure, sample size, and detection instrument parameters. Optimization depends not only on the nature of the sample, but also on the target species, or analyte. Detection of an analyte in blood can be very different from detecting the same analyte in urine in that the optimal conditions for high-pressure liquid chromatography (HPLC) fractionation may not be the same. The chromatography media that a skilled user selects may change according to the contents of the sample, and the solvents the skilled user selects need to be compatible with the ionization process for mass spectrometric analysis.
Commercial high-pressure liquid chromatography-mass spectrometry (HPLC-MS) systems may include two solvents (though one solvent of varying concentration can be used), a wash solvent and an elution solvent, being prepared for each analysis or batch of analysis. A skilled user prepares the solvents in concentrations appropriate for the sample and the selected chromatography media. It is typically the case that the ratio between the wash and elution solvents changes over time during fractionation, and in commercial systems, a skilled user may oversee this change in ratio between the solvents, which can also be thought of as a gradient in relative concentration in the solvents. The reason for this gradient, or change in ratio, in the solvents is that analytes and contaminants have differential affinity for the chromatography media as compared to the elution solvent. In most cases, analytes are preferentially bound onto the chromatography media in the presence of wash solvent, but as the relative amount of elution solvent is increased, the analyte will eventually become unbound from the chromatography media and flow out of the system with the elution solvent. The skilled high-pressure liquid chromatography-mass spectrometry (HPLC-MS) user understands the chemical properties of the analytes in each sample and selects appropriate high-pressure liquid chromatography (HPLC) conditions, including the wash and elution solvents and their relative concentration during fractionation, to obtain sufficient distinction between the contaminants in a sample and the desired analytes so that each can be detected.
The chromatographic columns used in commercial high-pressure liquid chromatography (HPLC) systems are quite large and designed for reuse. Since the media in the chromatographic columns are selected for different types of fractionation, multiple columns will often be associated with a commercial HPLC system. The size of the columns used in commercial HPLC systems can require a large amount of sample, as well as a large volume of solvents. Additionally, the intended long life-time of the chromatographic columns means that they are used multiple times, and so the skilled user, or analyst, needs to be cognizant of what the columns were used for in the past in order to determine the veracity of the results he or she obtains.